Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/102—Mutagenizing nucleic acids
  • C12N15/1024—In vivo mutagenesis using high mutation rate "mutator" host strains by inserting genetic material, e.g. encoding an error prone polymerase, disrupting a gene for mismatch repair
• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
  • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
  • C11D3/16—Organic compounds
  • C11D3/38—Products with no well-defined composition, e.g. natural products
  • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
  • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/21—Serine endopeptidases (3.4.21)
  • C12Y304/21062—Subtilisin (3.4.21.62)

Definitions

• the invention is in the field of enzyme technology.
• the invention relates to proteases whose amino acid sequence, in particular with a view to their use in detergents and cleaning agents, in particular with regard to liquid detergents and cleaning agents, have been changed in order to give them better storage stability and / or to improve their cleaning performance, and the nucleic acids coding for them and their production.
• the invention further relates to the uses of these proteases and processes in which they are used, and agents containing them, in particular detergents and cleaning agents, in particular liquid detergents and cleaning agents.
• proteases are among the most technically significant enzymes. They are the longest established enzymes for detergents and cleaning agents and are found in practically all modern, powerful detergents and cleaning agents. They break down protein-based soiling on the items to be cleaned. Among these, proteases of the subtilisin type (subtilases, subtilopeptidases, EC 3.4.21.62) are particularly important which are serine proteases due to the catalytically active amino acids. They act as non-specific endopeptidases and hydrolyze any acid amide bonds that are inside peptides or proteins. Their pH optimum is usually in the clearly alkaline range. An overview of this family can be found, for example, in the article " Subtilases: Subtilisin-like Proteases "by R.
• Subtilisin enzymes edited by R. Bott and C. Betzel, New York, 1996 .
• Subtilases are naturally formed by microorganisms. Among these, the subtilisins formed and secreted by Bacillus species should be mentioned as the most important group within the subtilases.
• proteases of the subtilisin type which are preferably used in washing and cleaning agents are the subtilisins BPN 'and Carlsberg, the protease PB92, the subtilisins 147 and 309, the alkaline protease from Bacillus lentus, in particular from Bacillus lentus DSM 5483, Subtilisin DY and the enzymes thermitase, proteinase K and the proteases TW3 and TW7, which can be assigned to the subtilases but no longer to the subtilisins in the narrower sense, and variants of the proteases mentioned which have an amino acid sequence which is different from the starting protease.
• Proteases are changed in a targeted or random manner by methods known from the prior art and are thus optimized, for example, for use in detergents and cleaning agents. This includes point, deletion or insertion mutagenesis or fusion with other proteins or protein parts. Correspondingly optimized variants are known for most proteases known from the prior art.
• proteases are suitable for use in liquid surfactant-containing preparations. Many proteases do not show sufficient catalytic performance in such preparations. For the use of proteases in detergents and cleaning agents, a high catalytic activity under conditions such as are present during a wash cycle and a high storage stability are particularly desirable.
• protease- and surfactant-containing liquid formulations from the prior art have the disadvantage that the proteases contained do not have satisfactory proteolytic activity under standard washing conditions (for example in a temperature range from 20 ° C. to 40 ° C.) or are not sufficiently stable in storage , and therefore the formulations do not show optimal cleaning performance on protease-sensitive soiling.
• the invention therefore relates to a protease comprising an amino acid sequence which has at least 70% sequence identity with the amino acid sequence given in SEQ ID NO: 1 over its entire length and, based on the numbering according to SEQ ID NO: 1, at (i) at least one of the positions which correspond to positions 3, 4, 99 or 199 has at least one amino acid substitution, and (ii) at least one of the positions which correspond to positions 9, 21, 42, 44, 105, 112, 113, 131, 137, 139, 141, 145, 159, 168, 176, 177, 182, 193, 198, 204, 205, 206, 210, 212, 230, 234, 250, 253, 255, 259 or 267, has at least one amino acid substitution.
• Another object of the invention is a method for producing a protease as defined above, which comprises introducing at least one amino acid substitution at (i) at least one of the positions which, based on the numbering according to SEQ ID NO: 1, positions 3, 4, 99 or 199, and (ii) at least one of the positions which, based on the numbering according to SEQ ID NO: 1, correspond to positions 9, 21, 42, 44, 105, 112, 113, 131, 137, 139, 141, 145, 159 , 168, 176, 177, 182, 193, 198, 204, 205, 206, 210, 212, 230, 234, 250, 253, 255, 259 or 267, into a starting molecule that has an amino acid sequence that is at least 70 % Sequence identity with the amino acid sequence given in SEQ ID NO: 1 over its entire length.
• a protease in the sense of the present patent application therefore includes both the protease as such and a protease produced by a method according to the invention. All statements on the protease therefore relate both to the protease as such and to the proteases produced by means of corresponding processes.
• nucleic acids coding for these proteases relate to the nucleic acids coding for these proteases, proteases according to the invention or non-human host cells containing nucleic acids and agents comprising proteases according to the invention, in particular detergents and cleaning agents, washing and cleaning methods, and uses of the proteases according to the invention in detergents or cleaning agents for removal of proteinaceous soiling.
• Numerical ranges that are specified in the format "from x to y" include the values mentioned. If multiple preferred numeric ranges are specified in this format, is it goes without saying that all areas resulting from the combination of the different endpoints are also covered.
• At least one as used herein means one or more, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more.
• Liquid as used herein includes liquids and gels as well as pasty compositions. It is preferred that the liquid compositions are flowable and pourable at room temperature, but it is also possible that they have a yield point.
• the present invention is based on the surprising finding of the inventors that amino acid substitutions at the positions described herein bring about improved storage stability and / or an improved cleaning performance of this modified protease in detergents and cleaning agents.
• the change (s) according to the invention leads to (i) at least one of the positions which correspond to positions 3, 4, 99 or 199 in relation to the numbering according to SEQ ID NO: 1, and (ii) at least one of the positions Positions that refer to the positions 9, 21, 42, 44, 105, 112, 113, 131, 137, 139, 141, 145, 159, 168, 176, 177, 182, 193 based on the numbering according to SEQ ID NO: 1 , 198, 204, 205, 206, 210, 212, 230, 234, 250, 253, 255, 259 or 267 correspond to an improved cleaning performance of this modified protease in detergents and cleaning agents on at least one protease-sensitive soiling.
• Proteases according to the invention consequently enable improved removal of at least one, preferably several, protease-sensitive soiling from textiles and / or hard surfaces, for example dishes.
• the change (s) according to the invention leads to (i) at least one of the positions which correspond to positions 3, 4, 99 or 199 in relation to the numbering according to SEQ ID NO: 1, and (ii) at least one of the positions Positions that refer to the positions 9, 21, 42, 44, 105, 112, 113, 131, 137, 139, 141, 145, 159, 168, 176, 177, 182, 193 based on the numbering according to SEQ ID NO: 1 , 198, 204, 205, 206, 210, 212, 230, 234, 250, 253, 255, 259 or 267 correspond to an improved storage stability of this modified protease in washing and cleaning agents.
• the protease instructs (i) at least one of the positions which correspond to positions 3, 4, 99 or 199, at least one amino acid substitution which is selected from the group consisting of 3T, 4I, 99E and 199I, and (ii) at least one of the positions corresponding to positions 9, 21, 42, 44, 105, 112, 113, 131, 137, 139, 141, 145, 159, 168, 176, 177, 182, 193, 198, 204, 205, 206, 210, 212, 230, 234, 250, 253, 255, 259 or 267 correspond to at least one amino acid substitution resulting from that of 9C, 21F, 21W, 42S, 42H, 44S, 105V, 112V, 113A, 131D, 137I, 139R, 141S, 145I, 159L, 168V, 176E, 177D, 182C , 193M, 198
• the protease according to the invention has (i) at least one of the positions which correspond to positions 3, 4, 99 or 199, at least one amino acid substitution which is selected from the group consisting of 3T, 4I, 99E and 199I, and (ii) at least one of the positions corresponding to positions 9, 21, 42, 44, 105, 112, 113, 131, 137, 139, 141, 145, 159, 168, 176, 177, 182, 193, 198, 204 , 205, 206, 210, 212, 230, 234, 250, 253, 255, 259 or 267 correspond to at least one amino acid substitution resulting from that of 9C, 21F, 21W, 42S, 42H, 44S, 105V, 112V, 113A , 131D, 137I, 139R, 141S, 145I, 159L, 168V, 176E, 177D, 182C, 193M, 198D, 204L,
• the protease according to the invention has (i) at least one of the positions which correspond to positions 3, 4, 99 or 199, at least one amino acid substitution which is selected from the group consisting of 3T, 4I, 99E and 199I, and (ii) at least one of the positions corresponding to positions 9, 21, 42, 44, 105, 112, 113, 131, 137, 139, 141, 145, 159, 168, 176, 177, 182, 193, 198, 204 , 205, 206, 210, 212, 230, 234, 250, 253, 255, 259 or 267 correspond to at least one amino acid substitution resulting from that of 9C, 21F, 21W, 42S, 42H, 44S, 105V, 112V, 113A , 131D, 137I, 139R, 141S, 145I, 159L, 168V, 176E, 177D, 182C, 193M, 198D, 204L,
• the protease according to the invention has (i) at least one of the positions which correspond to positions 3, 4, 99 or 199, at least one amino acid substitution which is selected from the group consisting of 3T, 4I, 99E and 199I, and (ii) at least one of the positions corresponding to positions 9, 21, 42, 44, 105, 112, 113, 131, 137, 139, 141, 145, 159, 168, 176, 177, 182, 193, 198, 204 , 205, 206, 210, 212, 230, 234, 250, 253, 255, 259 or 267 correspond to at least one amino acid substitution resulting from that of 9C, 21F, 21W, 42S, 42H, 44S, 105V, 112V, 113A , 131D, 137I, 139R, 141S, 145I, 159L, 168V, 176E, 177D, 182C, 193M, 198D, 204L,
• Certain embodiments of the proteases according to the invention have improved storage stability. They have an increased stability in detergents or cleaning agents compared to the wild-type enzyme (SEQ ID NO: 1) and in particular also to the starting variant of the protease (SEQ ID NO: 2) WO2013 / 060621A1 ), especially when stored for 3 or more days, 4 or more days, 7 or more days, 10 or more days, 12 or more days, 14 or more days, 21 or more days or 28 or more days.
• the proteases according to the invention can have an increased catalytic activity in detergents or cleaning agents, regardless of or in addition to the increased storage stability.
• the proteases according to the invention can have a proteolytic activity based on the wild type (SEQ ID NO: 1) and / or an already improved output variant of the protease (SEQ ID NO: 2) WO2013 / 060621A1 ) is at least 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109% or 110%.
• Such improved proteases enable improved washing results on proteolytically sensitive soiling in various temperature ranges, in particular a temperature range from 20 ° C. to 40 ° C.
• preferred embodiments of proteases according to the invention have particular stability in washing or cleaning agents, for example with respect to surfactants and / or bleaching agents and / or chelators, and / or with respect to temperature influences, in particular with respect to high temperatures, for example between 50 ° C. and 65 ° C., in particular 60 ° C, and / or against acidic or alkaline conditions and / or against pH changes and / or against denaturing or oxidizing agents and / or against proteolytic degradation and / or against a change in the redox ratios.
• performance-improved and / or temperature-stable protease variants are provided.
• cleaning performance is understood to mean the brightening performance on one or more soils, in particular on laundry or dishes.
• both the washing or cleaning agent which comprises the protease or the washing or cleaning liquor formed by this agent and the protease itself have a respective cleaning performance.
• the cleaning performance of the enzyme thus contributes to Cleaning performance of the agent or the washing or cleaning liquor formed by the agent.
• the cleaning performance is preferably determined as indicated below.
• the washing liquor is understood to be the use solution containing the washing or cleaning agent which acts on textiles or fabrics or hard surfaces and thus comes into contact with the soiling present on textiles or fabrics or hard surfaces.
• the washing liquor is usually formed when the washing or cleaning process begins and the washing or cleaning agent is diluted with water, for example in a dishwasher, a washing machine or in another suitable container.
• proteases of the invention have enzymatic activity, i.e. they are capable of hydrolysing peptides and proteins, especially in a washing or cleaning agent.
• a protease according to the invention is therefore an enzyme which catalyzes the hydrolysis of amide / peptide bonds in protein / peptide substrates and is therefore able to cleave proteins or peptides.
• a protease according to the invention is preferably a mature protease, i.e. around the catalytically active molecule without signal and / or propeptide (s). Unless otherwise stated, the sequences given also relate to mature (processed) enzymes.
• the protease is a free enzyme. This means that the protease can act directly with all components of an agent and, if the agent is a liquid agent, that the protease is in direct contact with the solvent of the agent (eg water).
• an agent may contain proteases that form an interaction complex with other molecules or that contain an "envelope".
• a single or several protease molecules can be separated from the other components of the agent by a structure surrounding them.
• a separating structure can result from, but is not limited to, vesicles, such as a micelle or a liposome.
• the surrounding structure can also be a virus particle, a bacterial cell or a eukaryotic cell.
• an agent can contain cells from Bacillus pumilus or Bacillus subtilus which express the proteases according to the invention, or cell culture supernatants of such cells.
• the protease comprises an amino acid sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77% over the entire length of the amino acid sequence given in SEQ ID NO: 1. , 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91, 5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5% , 98%, 98.5%, 98.8%, 99% and 99.5% is identical, and based on the numbering according to SEQ ID NO: 1 (i) at least one amino acid substitution in at least one of the positions which corresponds to the positions 3, 4, 99 or 199, wherein the at least one amino acid substitution is preferably from that of 3T, 4I, 99E and 199I
• the protease comprises an amino acid sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77% over the entire length of the amino acid sequence given in SEQ ID NO: 1. , 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91, 5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5% , 98%, 98.5%, 98.8%, 99% and 99.5% is identical, and based on the numbering according to SEQ ID NO: 1 (i) at least two amino acid substitutions in at least two of the positions that correspond to the positions 3, 4, 99 or 199, wherein the at least two amino acid substitutions are selected from the group consisting of 3T, 4I, 99E and
• the protease comprises an amino acid sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77% over the entire length of the amino acid sequence given in SEQ ID NO: 1. , 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91, 5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5% , 98%, 98.5%, 98.8%, 99% and 99.5% is identical, and based on the numbering according to SEQ ID NO: 1 (i) at least three amino acid substitutions in at least three of the positions that correspond to the positions 3, 4, 99 or 199, wherein the at least three amino acid substitutions are selected from the group consisting of 3T, 4I, 99E and
• the protease comprises an amino acid sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77 over the entire length of the amino acid sequence given in SEQ ID NO: 1 %, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91 , 5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5 %, 98%, 98.5%, 98.8%, 99% and 99.5% is identical, and based on the numbering according to SEQ ID NO: 1 (i) has the amino acid substitutions 3T, 4I, 99E and 199I, and (ii) at least one amino acid substitution at at least one of the positions which correspond to positions 9, 21, 42, 44, 105, 11
• a protease has at least one of the specified amino acid substitutions means that it contains at the respective position one (of the specified) amino acid substitution (s), i.e. at least the positions indicated are not otherwise mutated or deleted, for example by fragmentation of the protease.
• sequence comparison The identity of nucleic acid or amino acid sequences is determined by a sequence comparison. This sequence comparison is based on the BLAST algorithm established and commonly used in the prior art (cf. e.g. Altschul et al. (1990) "Basic local alignment search tool", J. Mol. Biol. 215: 403-410 , and Altschul et al. (1997): “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25: 3389-3402 ) and happens principally in that similar sequences of nucleotides or amino acids in the nucleic acid or amino acid sequences are assigned to each other. A tabular assignment of the relevant positions is called alignment.
• Sequence comparisons are created using computer programs.
• the Clustal series for example, are frequently used (cf. e.g. Chenna et al. (2003) "Multiple sequence alignment with the Clustal series of programs", Nucleic Acids Res. 31: 3497-3500 ), T-Coffee (see e.g. Notredame et al. (2000) "T-Coffee: A novel method for multiple sequence alignments", J. Mol. Biol. 302: 205-217 ) or programs based on these programs or algorithms.
• Sequence comparisons are also possible with the Vector NTI® Suite 10.3 computer program (Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California, USA) with the specified standard parameters, whose AlignX module for sequence comparisons is based on ClustalW. Unless otherwise stated, the sequence identity given herein is determined using the BLAST algorithm.
• Such a comparison also allows a statement to be made about the similarity of the compared sequences to one another. It is usually specified in percent identity, that is to say the proportion of identical nucleotides or amino acid residues in the same positions or in an alignment corresponding to one another.
• the broader concept of homology also includes conserved amino acid exchanges in amino acid sequences, that is, amino acids with similar chemical activity, since these usually have similar chemical activities within the protein.
• the similarity of the compared sequences can therefore also be given as percent homology or percent similarity.
• Identity and / or homology information can be made about entire polypeptides or genes or only over individual areas. Homologous or identical regions of different nucleic acid or amino acid sequences are therefore defined by matches in the sequences. Such areas often have identical functions.
• nucleic acid or amino acid sequence can be small and contain only a few nucleotides or amino acids. Such small areas often have essential functions for the overall activity of the protein. It can therefore make sense to relate sequence matches only to individual, possibly small, areas. Unless otherwise stated, identity or homology information in the present application relates to the total length of the nucleic acid or amino acid sequence specified in each case.
• the indication that an amino acid position corresponds to a numerically designated position in SEQ ID NO: 1 therefore means that the corresponding position is assigned to the numerically designated position in SEQ ID NO: 1 in an alignment as defined above.
• the protease is characterized in that its cleaning performance (after storage, for example over 3 weeks) compared to the wild-type enzyme (SEQ ID NO: 1) or one in WO201360621A1 described output variant is not significantly reduced, ie has at least 80% of the reference washing performance, preferably at least 100%, more preferably at least 110% or more.
• the cleaning performance can be determined in a washing system that contains a detergent in a dosage of between 4.5 and 7.0 grams per liter of washing liquor and the protease, the proteases to be compared being used with the same concentration (based on active protein) and the cleaning performance versus Soiling on cotton is determined by measuring the degree of cleaning of the washed textiles.
• the washing process can take place for 60 minutes at a temperature of 40 ° C and the water has a water hardness between 15.5 and 16.5 ° (German hardness).
• the concentration of the protease in the detergent intended for this washing system is 0.001 to 0.1% by weight, preferably 0.01 to 0.06% by weight, based on active, purified protein.
• a liquid reference detergent for such a washing system can, for example, be composed as follows (all figures in% by weight): 4.4% alkylbenzenesulfonic acid, 5.6% more anionic surfactants, 2.4% C 12 -C 18 Na salts of fatty acids (soaps), 4.4% non-ionic surfactants, 0.2% phosphonates, 1.4% citric acid, 0.95% NaOH, 0, 01% defoamer, 2% glycerin, 0.08% preservatives, 1% ethanol, remainder demineralized water.
• the dosage of the liquid detergent is preferably between 4.5 and 6.0 grams per liter of wash liquor, for example 4.7, 4.9 or 5.9 grams per liter of wash liquor. Washing is preferably carried out in a pH range between pH 7 and pH 10.5, preferably between pH 7.5 and pH 8.5.
• the cleaning performance is determined, for example, at 20 ° C or 40 ° C using a liquid detergent such as e.g. of the above, the washing process preferably being carried out for 60 minutes at 600 rpm.
• the degree of whiteness i.e. the lightening of the soiling, as a measure of the cleaning performance, is determined using optical measuring methods, preferably photometrically.
• a suitable device is, for example, the Minolta CM508d spectrometer.
• the devices used for the measurement are calibrated beforehand with a white standard, preferably a supplied white standard.
• a liquid reference hand dishwashing detergent for such a washing system can, for example, be composed as follows (all figures in% by weight): 8-20% alkylbenzenesulfonic acid, 30-80% demineralized water, 5.4% NaOH (50%), 7.14 % Fatty alcohol ether sulfate, 2.0% NaCl (20%), 0.383% phosphoric acid (H 3 PO 4 ; 34% / 85%), 0.1% preservative, 0.25% perfume, 1.0% dye, 0.04 % Bitter substance.
• the use of the respective protease with the same activity ensures that even if the ratio of active substance to total protein (the values of the specific activity) diverges, the respective enzymatic properties, for example the cleaning performance of certain soiling, are compared. In general, a low specific activity can be compensated for by adding a larger amount of protein.
• protease activity can be determined via the release of the chromophore para-nitroaniline (pNA) from the substrate suc-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (AAPF).
• pNA chromophore para-nitroaniline
• the protease cleaves the substrate and releases pNA.
• the release of the pNA causes an increase in the absorbance at 410 nm, the time course of which is a measure of the enzymatic activity (cf. Del Mar et al., 1979).
• the measurement is carried out at a temperature of 25 ° C, at pH 8.6 and a wavelength of 410 nm.
• the measurement time is 5 min and the measurement interval is 20s to 60s.
• the protease activity is usually in Protease units (PE) indicated. Suitable protease activities are, for example, 2.25, 5 or 10 PE per ml wash liquor. However, the protease activity is not zero.
• An alternative test for determining the proteolytic activity of the proteases according to the invention is an optical measurement method, preferably a photometric method.
• the test suitable for this involves the protease-dependent cleavage of the substrate protein casein.
• the protease breaks it down into a large number of smaller sub-products.
• the entirety of these partial products has an increased absorption at 290 nm compared to uncleaved casein, this increased absorption being determined using a photometer, and a conclusion being drawn on the enzymatic activity of the protease.
• the protein concentration can be determined using known methods, for example the BCA method (bicinchoninic acid; 2,2'-bichinolyl-4,4'-dicarboxylic acid) or the biuret method ( Gornall et al., J. Biol. Chem. 177 (1948): 751-766 ) can be determined.
• the determination of the active protein concentration can be carried out by titrating the active centers using a suitable irreversible inhibitor and determining the residual activity (cf. Bender et al., J. Am. Chem. Soc. 88, 24 (1966): 5890-5913 ) respectively.
• proteases according to the invention can have further amino acid changes, in particular amino acid substitutions, insertions or deletions.
• Such proteases are, for example, by targeted genetic modification, i.e. through mutagenesis processes, further developed and optimized for specific purposes or with regard to special properties (for example with regard to their catalytic activity, stability, etc.).
• nucleic acids according to the invention can be introduced into recombination approaches and thus used to generate completely novel proteases or other polypeptides.
• the aim is to introduce targeted mutations such as substitutions, insertions or deletions into the known molecules, for example in order to improve the cleaning performance of enzymes according to the invention.
• the surface charges and / or the isoelectric point of the molecules and thereby their interactions with the substrate can be changed.
• the net charge of the enzymes can be changed in order to influence the substrate binding, especially for use in detergents and cleaning agents.
• the stability or catalytic activity of the protease can be increased by one or more corresponding mutations and thereby its cleaning performance can be improved.
• Advantageous properties of individual mutations, for example individual substitutions can complement one another.
• a protease that has already been optimized with regard to certain properties, for example with regard to its stability during storage, can therefore be further developed within the scope of the invention.
• amino acid exchanges The following convention is used for the description of substitutions that relate to exactly one amino acid position (amino acid exchanges): First, the naturally present amino acid is designated in the form of the internationally used one-letter code, then the associated sequence position and finally the inserted amino acid. Multiple exchanges within the same polypeptide chain are separated from one another by slashes. In the case of insertions, additional amino acids are named after the sequence position. In the case of deletions, the missing amino acid is replaced by a symbol, for example an asterisk or a dash, or a ⁇ is given in front of the corresponding position.
• A95G describes the substitution of alanine at position 95 by glycine, A95AG the insertion of glycine after the amino acid alanine at position 95 and A95 * or ⁇ A59 the deletion of alanine at position 95.
• This nomenclature is known to the person skilled in the field of enzyme technology.
• Another object of the invention is therefore a protease, which is characterized in that it is obtainable from a protease as described above as the starting molecule by one or more conservative amino acid substitution, the protease being counted according to SEQ ID NO: 1 at least one of the amino acid substitutions described above.
• conservative amino acid substitution means the substitution of one amino acid residue for another amino acid residue, this exchange not leading to a change in polarity or charge at the position of the amino acid exchanged, e.g. the exchange of a non-polar amino acid residue for another non-polar amino acid residue.
• the protease is characterized in that it is obtainable from a protease according to the invention as a starting molecule by fragmentation, deletion, insertion or substitution mutagenesis and comprises an amino acid sequence which is at least 190, 200, 210, 220, 230, 240, 250, 260, 261, 262, 263, 264, 265, 266, 267, 268 or 269 connected amino acids coincides with the starting molecule, the protease (i) at least one amino acid substitution at at least one of the positions which corresponds to positions 3, 4, 99 or 199, and (ii) at least one amino acid substitution at at least one of the positions corresponding to positions 9, 21, 42, 44, 105, 112, 113, 131, 137, 139, 141, 145, 159, 168 , 176, 177, 182, 193, 198, 204, 205, 206, 210, 212, 230, 234, 250, 253, 255, 259 or 267.
• the enzymes retain their proteolytic activity even after the mutagenesis, ie their proteolytic activity corresponds at least that of the starting enzyme, ie in a preferred embodiment the proteolytic activity is at least 80%, preferably at least 90% of the activity of the starting enzyme.
• Other substitutions can also have beneficial effects. Both single and multiple contiguous amino acids can be exchanged for other amino acids.
• the amino acid positions are defined here by an alignment of the amino acid sequence of a protease according to the invention with the amino acid sequence of the protease from Bacillus lentus, as specified in SEQ ID NO: 1. Furthermore, the assignment of the positions depends on the mature (mature) protein. This assignment is also to be used in particular if the amino acid sequence of a protease according to the invention comprises a higher or lower number of amino acid residues than the protease from Bacillus lentus according to SEQ ID NO: 1. Starting from the positions mentioned in the amino acid sequence of the protease from Bacillus lentus , the change positions in a protease according to the invention are those which are assigned to these positions in an alignment.
• Advantageous positions for sequence changes, in particular substitutions, of the protease from Bacillus lentus which are preferably of importance when transferred to homologous positions of the proteases according to the invention and which impart advantageous functional properties to the protease, are accordingly the positions which correspond to the positions described here in an alignment, ie in the count according to SEQ ID NO: 1.
• the following amino acid residues are present in the wild-type molecule of the protease from Bacillus lentus (SEQ ID NO: 1): S3, V4, S9, L21, N42, R44, R99, I105, A112, G113, A131, V137, S139, T141 , V145, I159, A168, Q176, N177, S182, V193, N198, V199, P204, G205, S206, S210, N212, Q230, S234, S250, S253, N255, S259.
• Comparative tests can provide further confirmation of the correct assignment of the amino acids to be changed, that is to say in particular their functional correspondence, according to which the two positions assigned to one another on the basis of an alignment are changed in the same way in both proteases compared and it is observed whether both are present the enzymatic activity is changed in the same way.
• an amino acid exchange in a certain position of the protease from Bacillus lentus according to SEQ ID NO: 1 is accompanied by a change in an enzymatic parameter, for example an increase in the K M value, and a corresponding change in the enzymatic parameter, for example also a change
• an increase in the K M value, observed in a protease variant according to the invention, the amino acid exchange of which was achieved by the same introduced amino acid, is to be seen here as confirmation of the correct assignment.
• the protease or the protease produced using a method according to the invention is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92 , 5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5 %, 98.8%, 99% or 99.5% identical to the amino acid sequence given in SEQ ID NO: 1 over its entire length.
• the protease or the protease produced by a method according to the invention has (i) at least one of the 3T, 4I, 99E or 199I amino acid substitutions in at least one of the positions which correspond to positions 3, 4, 99 or 199, and (ii) at least one of amino acid substitutions 9C, 21F, 21W, 42S, 42H, 44S, 105V, 112V, 113A, 131D, 137I, 139R, 141S, 145I, 159L, 168V, 176E, 177D, 182C, 193M, 198D, 204L, 205D, 206A, 210C, 212D, 230E, 234A, 250N, 253C, 255Y, 259C or 267S in at least one of the positions that correspond to positions 9, 21, 42, 44, 105, 112, 113, 131, 137, 139, 141, 145, 159, 168, 176, 177, 182,
• Another object of the invention is a previously described protease which is additionally stabilized, in particular by one or more mutations, for example substitutions, or by coupling to a polymer.
• An increase in stability during storage and / or during use, for example in the washing process, means that the enzymatic activity lasts longer and thus the cleaning performance is improved.
• all stabilization options described and / or useful in the prior art come into consideration. Preference is given to those stabilizations which are achieved via mutations in the enzyme itself, since such stabilizations do not require any further steps following the extraction of the enzyme. Examples of suitable sequence changes are mentioned above. Further suitable sequence changes are known from the prior art.
• Preferred embodiments are those in which the enzyme is stabilized in several ways, since several stabilizing mutations have an additive or synergistic effect.
• Another object of the invention is a protease as described above, which is characterized in that it has at least one chemical modification.
• a protease with such a change is called a derivative, i.e. the protease is derivatized.
• derivatives are understood to mean those proteins whose pure amino acid chain has been chemically modified.
• derivatizations can be carried out, for example, in vivo by the host cell that expresses the protein. Couplings of low molecular weight compounds such as lipids or oligosaccharides are particularly noteworthy in this regard.
• derivatizations can also be carried out in vitro , for example by chemical conversion of a side chain of an amino acid or by covalent binding of another compound to the protein. For example, the coupling of amines to carboxyl groups of an enzyme to change the isoelectric point is possible.
• Such a different compound can also be a further protein which is bound to a protein according to the invention, for example via bifunctional chemical compounds.
• Derivatization is also to be understood to mean covalent binding to a macromolecular carrier or non-covalent inclusion in suitable macromolecular cage structures.
• derivatizations can influence the substrate specificity or the binding strength to the substrate, or can temporarily block the enzymatic activity if the coupled substance is an inhibitor. This can be useful, for example, for the period of storage.
• Such modifications can also affect stability or enzymatic activity. They can also serve to reduce the allergenicity and / or immunogenicity of the protein and thus, for example, to increase its skin tolerance.
• couplings with macromolecular compounds for example polyethylene glycol, can improve the protein with regard to stability and / or skin tolerance.
• Derivatives of a protein according to the invention can also be understood in the broadest sense to mean preparations of these proteins.
• a protein can be combined with various other substances, for example from the culture of the producing microorganisms.
• a protein may also have been specifically mixed with other substances, for example to increase its storage stability. All preparations of a protein according to the invention are therefore also according to the invention. This is also irrespective of whether it actually exhibits this enzymatic activity in a particular preparation or not. Because it may be desirable that it has little or no activity during storage and that it only develops its enzymatic function at the time of use. This can be controlled, for example, using appropriate accompanying substances. In particular, the joint preparation of proteases with specific inhibitors is possible in this regard.
• proteases or protease variants and / or derivatives described above those are particularly preferred within the scope of the present invention Storage stability and / or its cleaning performance is improved compared to the initial variant, the cleaning performance in a washing system being determined as described above.
• Another object of the invention is a nucleic acid which codes for a protease according to the invention, and a vector containing such a nucleic acid, in particular a cloning vector or an expression vector.
• RNA molecules can be DNA or RNA molecules. They can be present as a single strand, as a single strand complementary to this single strand or as a double strand. In the case of DNA molecules in particular, the sequences of both complementary strands must be taken into account in all three possible reading frames. It should also be taken into account that different codons, ie base triplets, can code for the same amino acids, so that a certain amino acid sequence can be encoded by several different nucleic acids. Because of this degeneracy of the genetic code, all nucleic acid sequences which can encode one of the proteases described above are included in this subject matter of the invention.
• nucleic acid sequences without any doubt since, despite the degeneracy of the genetic code, individual codons can be assigned to defined codons. The person skilled in the art can therefore easily determine nucleic acids coding for this amino acid sequence on the basis of an amino acid sequence. Furthermore, one or more codons can be replaced by synonymous codons in the nucleic acids according to the invention.
• This aspect relates in particular to the heterologous expression of the enzymes according to the invention. Every organism, for example a host cell of a production strain, has a certain codon usage. Codon use means the translation of the genetic code into amino acids by the respective organism.
• Bottlenecks in protein biosynthesis can occur if the codons lying on the nucleic acid in the organism are opposed to a comparatively small number of loaded tRNA molecules. Although coding for the same amino acid, this means that a codon in the organism is translated less efficiently than a synonymous codon that codes for the same amino acid. Due to the presence of a higher number of tRNA molecules for the synonymous codon, this can be translated more efficiently in the organism.
• vectors are understood to mean elements consisting of nucleic acids which contain a nucleic acid according to the invention as the characteristic nucleic acid region. They are able to establish this in a species or a cell line as a stable genetic element over several generations or cell divisions.
• Vectors are special plasmids, in particular circular genetic elements, when used in bacteria.
• a nucleic acid according to the invention is cloned into a vector.
• the vectors include, for example, those whose origin is bacterial plasmids, viruses or bacteriophages, or predominantly synthetic vectors or plasmids with elements of very different origins. With the other genetic elements present in each case, vectors can establish themselves as stable units in the host cells concerned over several generations. They can be extrachromosomal as separate units or integrated into a chromosome or chromosomal DNA.
• Expression vectors comprise nucleic acid sequences which enable them to replicate in the host cells, preferably microorganisms, particularly preferably bacteria, containing them and to express the nucleic acid contained therein. Expression is influenced in particular by the promoter or promoters which regulate the transcription. In principle, expression can take place through the natural promoter originally located before the nucleic acid to be expressed, but also through a promoter of the host cell provided on the expression vector or also through a modified or a completely different promoter from another organism or another host cell. In the present case, at least one promoter is provided for the expression of a nucleic acid according to the invention and used for its expression.
• Expression vectors can also be regulated, for example by changing the cultivation conditions or when a specific cell density of the host cells containing them has been reached or by adding certain substances, in particular activators of gene expression.
• An example of such a substance is the galactose derivative isopropyl- ⁇ -D-thiogalactopyranoside (IPTG), which is used as an activator of the bacterial lactose operon (lac operon).
• IPTG galactose derivative isopropyl- ⁇ -D-thiogalactopyranoside
• lac operon lac operon
• the invention further relates to a non-human host cell which contains a nucleic acid or a vector according to the invention or which contains a protease according to the invention, in particular one which secretes the protease into the medium surrounding the host cell.
• a nucleic acid according to the invention or a vector according to the invention is preferably transformed into a microorganism which then represents a host cell according to the invention.
• individual components, ie nucleic acid parts or fragments of a nucleic acid according to the invention can also be introduced into a host cell in such a way that the resulting host cell contains a nucleic acid or a vector according to the invention.
• This procedure is particularly suitable if the host cell already has one or more components of one contains nucleic acid according to the invention or a vector according to the invention and the further constituents are then supplemented accordingly.
• Methods for transforming cells are established in the prior art and are well known to the person skilled in the art. In principle, all cells are suitable as host cells, that is to say prokaryotic or eukaryotic cells.
• host cells that can be genetically advantageously handled, for example in relation to the transformation with the nucleic acid or the vector and its stable establishment, for example unicellular fungi or bacteria.
• Preferred host cells are furthermore distinguished by good microbiological and biotechnological manageability.
• Preferred host cells according to the invention secrete the (transgenic) expressed protein into the medium surrounding the host cells.
• the proteases can be modified by the cells producing them after their production, for example by attaching sugar molecules, formylations, aminations, etc. Such post-translational modifications can functionally influence the protease.
• Further preferred embodiments are those host cells whose activity can be regulated on the basis of genetic regulatory elements which are provided, for example, on the vector, but which may also be present in these cells from the outset. For example, by the controlled addition of chemical compounds that serve as activators, by changing the cultivation conditions or when a certain cell density is reached, these can be stimulated for expression. This enables economical production of the proteins according to the invention.
• An example of such a connection is IPTG as described above.
• Preferred host cells are prokaryotic or bacterial cells.
• Bacteria are characterized by short generation times and low demands on the cultivation conditions. As a result, inexpensive cultivation processes or production processes can be established.
• the specialist in bacteria in fermentation technology has a wealth of experience.
• Gram-negative or gram-positive bacteria can be suitable for a special production for various reasons, which can be determined experimentally in individual cases, such as nutrient sources, product formation rate, time requirement etc.
• Gram-negative bacteria such as Escherichia coli
• a large number of proteins are secreted into the periplasmic space, i.e. into the compartment between the two membranes that enclose the cells.
• Gram-negative bacteria can also be designed in such a way that they not only express the expressed proteins into the periplasmic space, but also into the medium surrounding the bacteria.
• Gram-positive bacteria such as Bacilli or Actinomycetes or other representatives of the Actinomycetales , on the other hand, have no outer membrane, so that secreted proteins are immediately released into the medium surrounding the bacteria, usually the nutrient medium from which the expressed proteins can be purified. They can be isolated directly from the medium or processed further.
• gram-positive bacteria are related or identical to most organisms of origin for technically important enzymes and usually form comparable enzymes themselves, so that they have a similar codon use and their protein synthesis apparatus is naturally designed accordingly.
• Host cells according to the invention can be changed with regard to their requirements for the culture conditions, have different or additional selection markers or can also express other or additional proteins.
• these can also be host cells which transgenically express several proteins or enzymes.
• the present invention is in principle applicable to all microorganisms, in particular to all fermentable microorganisms, particularly preferably to those of the genus Bacillus , and leads to the fact that proteins of the invention can be produced by using such microorganisms. Such microorganisms then represent host cells in the sense of the invention.
• the host cell is characterized in that it is a bacterium, preferably one which is selected from the group of the genera of Escherichia, Klebsiella, Bacillus, Staphylococcus, Corynebacterium, Arthrobacter, Streptomyces, Stenotrophomonas and Pseudomonas , more preferably one that is selected from the group of Escherichia coli, Klebsiella planticola, Bacillus licheniformis, Bacillus lentus, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus alcalophilus, Bacillus globigii, Bacillus gibsonii, Bacillus clausii, Bacillus halodurans, Bacillus pumilus, Staphylococcus carnosus, Corynebacterium glutamicum , Arthrobacter oxidans , Streptomyces lividans , Streptomy
• the host cell can also be a eukaryotic cell, which is characterized in that it has a cell nucleus.
• Another object of the invention is therefore a host cell, which is characterized in that it has a cell nucleus.
• eukaryotic cells are able to post-translationally modify the protein formed. Examples include fungi such as Actinomycetes or yeasts such as Saccharomyces or Kluyveromyces . This can be particularly advantageous, for example, if the proteins are to undergo specific modifications in connection with their synthesis which enable such systems.
• modifications that eukaryotic systems carry out in particular in connection with protein synthesis include, for example, the binding of low molecular weight compounds such as membrane anchors or oligosaccharides. Such oligosaccharide modifications may be desirable, for example, to reduce the allergenicity of an expressed protein. Co-expression with the enzymes naturally formed by such cells, such as cellulases, can also be advantageous. Furthermore, for example, thermophilic fungal expression systems are particularly suitable for the expression of temperature-resistant proteins or variants.
• the host cells according to the invention are cultivated and fermented in a conventional manner, for example in discontinuous or continuous systems.
• a suitable nutrient medium is inoculated with the host cells and the product is harvested from the medium after an experimentally determined period.
• Continuous fermentations are characterized by achieving a steady state in which cells partially die but also regrow over a comparatively long period of time and at the same time the protein formed can be removed from the medium.
• This subject of the invention preferably comprises fermentation processes. Fermentation processes are known per se from the prior art and represent the actual large-scale production step, usually followed by a suitable purification method of the product produced, for example the proteases according to the invention. All fermentation processes which are based on a corresponding process for producing a protease according to the invention represent embodiments of this subject of the invention.
• Fermentation processes which are characterized in that the fermentation is carried out via a feed strategy, are particularly suitable.
• the media components that are consumed by the ongoing cultivation are fed.
• considerable increases can be achieved both in the cell density and in the cell mass or dry mass and / or in particular in the activity of the protease of interest.
• the fermentation can also be designed in such a way that undesired metabolic products are filtered out or neutralized by adding buffer or suitable counterions.
• the protease produced can be harvested from the fermentation medium.
• Such a fermentation method is preferred over isolation of the protease from the host cell, ie product preparation from the cell mass (dry mass), but requires the provision of suitable host cells or one or more suitable secretion markers or mechanisms and / or transport systems so that the host cells can Secrete the protease into the fermentation medium.
• the isolation of the Protease from the host cell ie a purification of the same from the cell mass, is carried out, for example by precipitation with ammonium sulfate or ethanol, or by chromatographic purification.
• Another object of the invention is an agent which is characterized in that it contains a protease according to the invention as described above.
• the agent is preferably a washing or cleaning agent.
• washing or cleaning agents both concentrates and agents to be used undiluted, for use on a commercial scale, in the washing machine or for hand washing or cleaning.
• detergents for textiles, carpets, or natural fibers, for which the term detergent is used.
• dishwashing detergents for dishwashers or manual dishwashing detergents or cleaners for hard surfaces such as metal, glass, porcelain, ceramics, tiles, stone, painted surfaces, plastics, wood or leather, for which the term cleaning agent is used, i.e. in addition to manual and machine Dishwashing detergents, for example, also scouring agents, glass cleaners, toilet scent detergents, etc.
• detergents and cleaning agents in the context of the invention also include washing aids which are added to the actual detergent in the case of manual or machine textile washing in order to achieve a further effect.
• detergents and cleaning agents within the scope of the invention also include textile pretreatment and post-treatment agents, i.e. agents with which the item of laundry is brought into contact before the actual laundry, for example for dissolving stubborn soiling, and also agents which are contained in one of the Actual textile laundry downstream step give the laundry further desirable properties such as a comfortable grip, wrinkle-free or low static charge. The latter means, among others, the fabric softener counted.
• the detergents or cleaning agents according to the invention which can be present as powdery solids, in post-compacted particle form, as gels, homogeneous solutions or suspensions, can contain, in addition to a protease according to the invention, all known ingredients customary in such agents, preferably at least one further ingredient in the agent is available.
• the agents according to the invention can in particular contain surfactants, builders (builders), peroxygen compounds or bleach activators. Furthermore, they can contain water-miscible organic solvents, further enzymes, sequestering agents, electrolytes, pH regulators and / or further auxiliaries such as optical brighteners, graying inhibitors, foam regulators as well as colors and fragrances and combinations thereof.
• a combination of a protease according to the invention with one or more further ingredient (s) of the agent is advantageous, since such agent in preferred embodiments according to the invention has an improved cleaning performance due to the resulting synergisms.
• Such a synergism can be achieved in particular by combining a protease according to the invention with a surfactant and / or a builder (builder) and / or a peroxygen compound and / or a bleach activator.
• the agent according to the invention cannot contain boric acid.
• An agent according to the invention advantageously contains the protease in an amount of 2 ⁇ g to 20mg, preferably 5 ⁇ g to 17.5mg, particularly preferably 20 ⁇ g to 15mg and very particularly preferably 50 ⁇ g to 10mg per g of the agent.
• the concentration of the protease (active enzyme) described herein in the agent is> 0 to 1% by weight, preferably 0.001 to 0.1% by weight, based on the total weight of the agent or the composition.
• the protease and / or further ingredients of the agent contained in the agent can be coated with a substance which is impermeable to the enzyme at room temperature or in the absence of water, which substance becomes permeable to the enzyme under conditions of use of the agent.
• Such an embodiment of the invention is thus characterized in that the protease is coated with a substance impermeable to the protease at room temperature or in the absence of water.
• the washing or cleaning agent itself can also be packaged in a container, preferably an air-permeable container, from which it is released shortly before use or during the washing process.
• inventions of the present invention include all solid, powder, liquid, gel or pasty dosage forms of agents according to the invention, which can optionally also consist of several phases and can be in compressed or uncompressed form.
• the agent can be in the form of a free-flowing powder, in particular with a bulk density of 300 g / l to 1200 g / l, in particular 500 g / l to 900 g / l or 600 g / l to 850 g / l.
• the solid dosage forms of the agent also include extrudates, granules, tablets or pouches.
• the agent can also be liquid, gel-like or pasty, for example in the form of a non-aqueous liquid detergent or a non-aqueous paste or in the form of an aqueous liquid detergent or a water-containing paste.
• Liquid funds are generally preferred.
• the agent can be in the form of a one-component system. Such funds consist of one phase.
• an agent can consist of several phases. Such an agent is therefore divided into several components.
• Washing or cleaning agents according to the invention can only contain a protease. Alternatively, they can also contain further hydrolytic enzymes or other enzymes in a concentration appropriate for the effectiveness of the agent. A further embodiment of the invention thus represent agents which further comprise one or more further enzymes.
• All enzymes which can develop a catalytic activity in the agent according to the invention in particular a lipase, amylase, cellulase, hemicellulase, mannanase, tannase, xylanase, xanthanase, xyloglucanase, ⁇ -glucosidase, pectinase, carrageenase, perhydrolase, can preferably be used as further enzymes.
• Oxidase, oxidoreductase or another protease - distinguishable from the proteases according to the invention - and mixtures thereof.
• Further enzymes are advantageously contained in the agent in each case in an amount of 1x10 -8 to 5% by weight, based on active protein.
• Each additional enzyme is increasingly preferred in an amount of 1x10 -7 to 3% by weight, from 0.00001 to 1% by weight, from 0.00005 to 0.5% by weight, from 0.0001 to 0 , 1 wt .-% and particularly preferably from 0.0001 to 0.05 wt .-% in agents according to the invention, each based on active protein.
• the enzymes particularly preferably show synergistic cleaning performance against certain soiling or stains, ie the enzymes contained in the composition of the agents mutually support one another in their cleaning performance.
• Such a synergism is very particularly preferably present between the protease contained according to the invention and a further enzyme of an agent according to the invention, including in particular between said protease and an amylase and / or a lipase and / or a mannanase and / or a cellulase and / or a pectinase .
• Synergistic effects can occur not only between different enzymes, but also between one or more enzymes and other ingredients of the agent according to the invention.
• the enzymes to be used can also be packaged together with accompanying substances, for example from fermentation.
• the enzymes are preferably used as an enzyme liquid formulation (s).
• the enzymes are not provided in the form of the pure protein, but rather in the form of stabilized, storable and transportable preparations.
• These prefabricated preparations include, for example, the solid preparations obtained by granulation, extrusion or lyophilization or, in particular in the case of liquid or gel form agents, Solutions of the enzymes, advantageously concentrated as possible, low in water and / or mixed with stabilizers or other auxiliaries.
• the enzymes can be encapsulated both for the solid and for the liquid administration form, for example by spray drying or extrusion of the enzyme solution together with a preferably natural polymer or in the form of capsules, for example those in which the enzymes are enclosed as in a solidified gel or in those of the core-shell type in which an enzyme-containing core is coated with a protective layer impermeable to water, air and / or chemicals.
• Additional active ingredients for example stabilizers, emulsifiers, pigments, bleaching agents or dyes, can additionally be applied in superimposed layers.
• Capsules of this type are applied by methods known per se, for example by shaking or roll granulation or in fluid-bed processes. Such granules are advantageously low in dust, for example by applying polymeric film formers, and are stable on storage due to the coating.
• water-soluble films as are used, for example, in the formulation of detergents and cleaning agents in unit dosage form.
• Such a film enables the enzymes to be released after contact with water.
• water soluble refers to a film structure that is preferably completely water soluble.
• Such a film preferably consists of (fully or partially hydrolyzed) polyvinyl alcohol (PVA).
• Another object of the invention is a method for cleaning textiles or hard surfaces, which is characterized in that an agent according to the invention is used in at least one process step, or that a protease according to the invention becomes catalytically active in at least one process step, in particular in such a way that the protease in an amount of 40 ⁇ g to 4g, preferably from 50 ⁇ g to 3g, particularly preferably from 100 ⁇ g to 2g and very particularly preferably from 200 ⁇ g to 1g or in the concentrations described herein.
• the method described above is characterized in that the protease is used at a temperature of 0 to 100 ° C., preferably 0 to 60 ° C., more preferably 20 to 40 ° C. and most preferably 25 ° C. becomes.
• Processes for cleaning textiles are generally characterized in that different cleaning-active substances are applied to the in several process steps Items to be cleaned are applied and washed off after the exposure time, or that the items to be cleaned are treated in some other way with a detergent or a solution or dilution of this agent.
• a detergent or a solution or dilution of this agent is applied to processes for cleaning all materials other than textiles, especially hard surfaces. All conceivable washing or cleaning processes can be enriched in at least one of the process steps by the use of a washing or cleaning agent according to the invention or a protease according to the invention and then represent embodiments of the present invention.
• proteases according to the invention naturally already have a hydrolytic activity and also develop this in media which otherwise have no cleaning power, such as, for example, in mere buffer, a single and / or the only step of such a method can consist in the protease according to the invention being the only active cleaning component is brought into contact with the soiling, preferably in a buffer solution or in water. This represents a further embodiment of this subject matter of the invention.
• Alternative embodiments of this subject of the invention also represent methods for treating textile raw materials or for textile care, in which a protease according to the invention becomes active in at least one method step.
• processes for textile raw materials, fibers or textiles with natural components are preferred, and very particularly for those with wool or silk.
• the invention also covers the use of the proteases described herein in detergents or cleaning agents, for example as described above, for (improved) removal of protein-containing soiling, for example from textiles or hard surfaces.
• the protease in the washing or cleaning agent is 3 or more days, 4 or more days, 7 or more days, 10 or more days, 12 or more days, 14 or more days, 21 before a washing or cleaning process or stored for more days or 28 or more days.
• the invention relates to an alkaline protease of the subtilisin type from Bacillus lentus .
• an initial variant protease according to SEQ ID NO: 2 WO2013 / 060621A1
• variants were produced by random mutagenesis, which were then screened, inter alia, for improved washing performance and / or enzyme stability. In this way, 27 mutants with improved storage stability and / or improved cleaning performance were generated from the protease mentioned.
• the protease variants according to the invention were expressed in Bacillus subtilis .
• the proteases were diluted to the same activity level from the supernatants of the Bacillus subtilis culture.
• 50% detergent matrix without boric acid or 50% detergent matrix with 50% appropriately diluted Bacillus subtilis culture was added and mixed well.
• the sealed vessels were stored at 40 ° C for three and 3.5 weeks, respectively.
• the sample amount removed was dissolved in 0.1M Tris / HCl (pH 8.6) for 20 min at room temperature by stirring.
• the AAPF assay was then performed as described below.
• the activity of the protease is determined by the release of the chromophore para-nitroaniline from the substrate succinyl alanine-alanine-proline-phenylalanine-para-nitroanilide (AAPFpNA; Bachem L-1400).
• AAPFpNA succinyl alanine-alanine-proline-phenylalanine-para-nitroanilide
• the measurement was carried out at a temperature of 25 ° C., pH 8.6 and a wavelength of 410 nm.
• the measurement time was 5 minutes with a measurement interval of 20 to 60 seconds.
• variant Improved stability compared to the stability of the starting variant after 3 weeks of storage at 40 ° C. in the above-mentioned detergent or dishwashing matrix is shown below: variant Improved stability (%) in the above detergent matrix Improved stability (%) in the above-mentioned dishwasher detergent matrix
• variant Improved stability % in the above detergent matrix
• Improved stability % in the above-mentioned dishwasher detergent matrix
• Variants 10 and 13 show increased washing performance compared to the original variant.
• Proteases according to the invention in particular variants 10 and 13, consequently not only show improved storage stability, but also improved cleaning performance.

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